Advantages

The primary advantage of discontinuous buffer systems is the ability to concentrate the sample zone. The passage of the moving boundary has the effect of sweeping the sample into an extremely thin starting band. For analytical applications this can lead to reduced band widths and higher resolution separations. This feature has obvious advantages for the characterization of closely migrating species. For preparative applications, the result is the concentration of dilute samples. Another advantage of discontinuous buffer systems is their use as an analytical tool for defining relative mobilities. The mobility of the ion front is easily defined and can serve as a reference for defining relative mobilities. Furthermore, the mobility of the front is reproducible and independent of the gel matrix. These features can be convenient for analytical and forensic applications. Additionally, the mobility of sample ions can be altered by changing the mobility of ions in the trailing zone. This can lead to the tailoring of separations by defining the size range that can be fractionated. Sample ions that are not of interest can remain trapped in an ion front, allowing examination of particular ions. Finally, discontinuous buffer systems are compatible with virtually any gel format, as its use is independent of the gel matrix or the physical format of the gel. Since it alters only the buffer system, the only complication is selecting an effective buffer. Fortunately, the characteristics of many buffer systems have been described and designing new systems is a straightforward task, as described below.

Figure 2 exemplifies some of the features of discontinuous buffer systems for nucleic acid separations. Shown here is a portion of a DNA sequencing gel using a denaturing formate-glycine discontinuous buffer system. An intermediate zone, with a mobility between the formate leading ion and the glycine trailing ion, was inadvertently created from contaminating ions in the gel or sample loading buffer. For analytical application, care should be taken to ensure that extraneous ions are absent. However, the presence of this ion species demonstrates the effects of stacking limits on the size selection and band concentration of DNA sequencing products. DNA bands smaller than 106 bases are trapped in the first ion front while DNA sizes 115-166 are trapped in the second front. The DNA sizes that are trapped in the ion fronts define the stacking limits and cannot be separated because their mobility is between that of the migrating ion zones. Since the DNA is not freely electrophoresing, these stacking limits could be altered by changes to the gel matrix. The intermediate ion zone demonstrates other features. Its size is directly proportional to the amount of contaminating ion that is present. Larger amounts of contaminating ion would expand this zone. Additionally, the eight DNA bands in this zone are much sharper than the bands migrating behind the second front, even after the 30 cm migration distance. This is due to the concentrating effects of the leading ion front and the closely matched mobility between these DNA bands and the ions in the zone.

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